Thieno[3,2-d]pyrimidine derivative compound having inhibitory activity for protein kinase

ABSTRACT

The present invention relates to a compound, 4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide, and pharmaceutically acceptable salts thereof having inhibitory activity for protein kinases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 16/768,538filed on May 29, 2020, which is a U.S. National Phase Application ofPCT/KR2018/014997, filed Nov. 29, 2018, which claims the benefit ofpriority to U.S. Provisional Application Ser. No. 62/592,679 filed onNov. 30, 2017, the contents of which are hereby expressly incorporatedby reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to a thieno[3,2-d]-pyrimidine compound,4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide,and pharmaceutically acceptable salts thereof having inhibitory activityfor protein kinases, and a pharmaceutical composition comprising thesame as an active ingredient for the treatment of diseases caused byabnormal cell growth.

BACKGROUND OF THE INVENTION

A protein kinase is an enzyme which plays a key role in mediating signaltransduction via phosphorylation of a hydroxyl group present in atyrosine, serine or threonine residue, and, thus, is involved in theregulation of cell growth, differentiation, and proliferation.

As is well known, a balance between “on-states” and “off-states” of anintracellular signaling pathway is essential for maintenance ofhomeostasis of a cell. When a normal intracellular signaling pathway of,e.g., mostly continuation of “on-state” of intracellular signals isinterrupted due to overexpression or mutation of a specific proteinkinase, it may lead to an outbreak of various diseases such as cancer,inflammatory disease, metabolic disease and brain disease. It isestimated that human genome contains 518 protein kinases whichconstitute approximately 1.7% of all human genes [Manning et al.,Science, 298, (2002), 1912]. The protein kinases can be divided intotyrosine protein kinases (90 or more types) and serine/threonine proteinkinases. The tyrosine protein kinases can be divided into receptortyrosine kinases including 58 distinct kinases which can be furthercategorized into 20 subtypes, and cytoplasmic/non-receptor tyrosinekinases including 32 distinct kinases which can be further categorizedinto 10 subtypes. A receptor tyrosine kinase has a kinase domain on thesurface where it can bind a growth factor, and an active site wherephosphorylation of a tyrosine residue takes place. Binding of a growthfactor to the extracellular domain of the receptor may cause thereceptor tyrosine kinase to form a polymer, which may result inautophosphorylation of specific tyrosine residues in the cytoplasmicdomain. This may trigger, a cascade of events through phosphorylation ofintracellular proteins that ultimately transmit the extracellular signalto the nucleus, thereby causing transcription and synthesis of variousgenes that may be involved in cell growth, differentiation,proliferation and the like.

Among the various cytoplasmic kinases, RAF is one of the kinases thatparticipate in the linear Ras-RAF-MEK-ERK mitogen-activated proteinkinase (MAPK) pathway initiated by a receptor protein kinase, which isactivated by a growth factor [Solit, D. B. et al., Nature, 439, (2006),358]. There are known three types of RAF isoforms, A-RAF, B-RAF andC-RAF (RAF-1) [Jansen H W, et al., EMBO J, 2, (1983), 1969; Marais R. etal., Cancer Surv, 27, (1996), 101]. Since abnormal activation in theMAPK pathway has been observed in approximately 30% of human cancertissues and gene mutation of B-RAF and C-RAF showing aberrant activationhas been confirmed in cancer tissues, it is generally accepted that RAFplays a very important role in the MAPK pathway of cancer tissues.

Accordingly, there have been suggested methods of using a compoundhaving an inhibitory effect against abnormal activities of RAF kinasesfor treatment of cancer. Hence, a number of RAF and modified RAF kinaseinhibitors are currently under development or being tested in ongoingclinical studies. Examples of such RAF kinase inhibitors includesorafenib (Nexavar®, Bayer) which is used for treatment of liver cancer,and vemurafenib (ZELBORAF®, PLX-4032, RG7204, Roche) which has beenapproved for treatment of melanoma. Other examples of RAF kinaseinhibitors that are currently being tested in clinical trials include:regorafenib and RDEA119 by Bayer; RAF265 by Novartis; E3810 by AdvanChem; DCC2036 by Deciphera Pharma.; CKI-27 by Chugai Pharma.; andRO-5126766 by Roche.

However, efficacy of such drugs has been questioned when they areadministered over a duration of time despite their good initialperformance, as drug resistance has been observed in some patients about7 months after the initial administration of the drug.

It has been postulated that such degradation may be due to the drugresistance of B-RAF inhibitors, which is caused by abnormal activationof the MAPK pathway due to changes in RAF, activation of complementarysignaling systems among different RAF isoforms, or activation of variousreceptor kinases other than MAPK as a result of activation of differentpathways of Ras, a key protein used in the signal-transducing cascadewhich consists of K-Ras, N-Ras and H-Ras subtypes.

One of the signaling pathways that does not involve RAF kinases is C-FMS(cellular feline McDonough sarcoma), also known as colony-stimulatingfactor-1 receptor (CSF-1R), which is a member of the family of genesoriginally isolated from the Susan McDonough strain of feline sarcomaviruses. FMS is a receptor for macrophage-colony-stimulating factor(M-CSF) encoded by the C-FMS proto-oncogene, which belongs to a classIII RTK along with Kit, Flt-3 and PDGFR. It has been reported that FMStyrosine kinase is involved in cancer metastasis.

Another example is a receptor protein tyrosine kinase called discoidindomain receptor (DDR), which is a subfamily of receptor tyrosine kinasesthat possess an extracellular domain related to the lectin discoidin. Incase of animals such as humans, there are two types of DDR proteins,DDR1 type and DDR2 type, which have similar amino acid sequences, butthat are encoded by different genes. It has been reported that DDRproteins may be implicated in the process of cancer growth andmetastasis. In addition, an upregulated expression of DDR has beenobserved in some tumor cells, along with a report that an upregulatedexpression of DDR raised expression of MMP-1 and MMP-2 which are knownto be implicated in cancer growth. Thus, it is believed that inhibitionof such kinases can lead to a therapeutic effect against various typesof cancer.

Compounds having improved protein kinase inhibitory activity aredisclosed in International Publication Number WO 2013/100632.

DISCLOSURE OF INVENTION Technical Problem

There is a continuing need in the art for a compound having inhibitoryactivity against RAF, FMS, and DDR1 and DDR2 kinases and that providesfor improved treatment of various cancers including drug-resistantcancer, as compared with a conventional RAF kinase inhibitor.

Solution to Problem

Accordingly, the present invention provides a compound and apharmaceutical composition comprising same having efficaciouspharmacokinetic and pharmacodynamic properties for prevention ortreatment of intractable cancer, including drug-resistant cancer, byinhibiting RAF (a key regulator of cell growth, differentiation andproliferation), FMS, and DDR1 and DDR2 kinases.

One embodiment of the present invention relates to a compound,4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide,of formula (I), or a pharmaceutically acceptable salt, or hydratethereof:

The term “salt or hydrate thereof”, and variations thereof, as usedherein in reference to the compound of formula (I), encompasses salts ofthe compound of formula (I), hydrates of the compound of formula (I),and hydrates of salts of the compound of formula (I).In some embodiments, the compound of formula (I), or a pharmaceuticallyacceptable salt or hydrate thereof, has a purity of at least 95.0%.

In any of the various embodiments, the compound of formula (I), andpharmaceutically acceptable salts and hydrates thereof, have inhibitoryactivity against protein kinases.

In one embodiment, the inhibitory activity is against one or more ofA-RAF, B-RAF, C-RAF, DDR1, DDR2 and FMS.

In one embodiment, formula (I) is a pan-RAF inhibitor. In suchembodiments, the inhibitory activity is against A-RAF, B-RAF and C-RAF.

In another embodiment, the inhibitory activity is against FMS.

In still another embodiment, the activity is against DDR1 and DDR2kinases.

Another aspect of the present invention also relates to a pharmaceuticalcomposition comprising the compound of formula (I), or apharmaceutically acceptable salt or hydrate thereof.

Another aspect of the present invention also relates to methods forprevention or treatment of a disease mediated by abnormal activation ofone or more protein kinases, the method comprising administering any ofthe compounds or compositions comprising said compounds of the presentinvention to a mammal in need thereof.

Advantageous Effects of Invention

The inventive compound,4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidehaving inhibitory activity for protein kinases, can effectively inhibitvarious protein kinases including RAF, and thus can be used, singly orin combination, for prevention and treatment of diseases associated withaberrant cell growth which are caused by mutation or overexpression ofRAS protein or overactivation of its associated protein kinases.

MODE FOR THE INVENTION

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents which may be included within the scope ofthe present invention as defined by the claims. One skilled in the artwill recognize many methods and materials similar or equivalent to thosedescribed herein, which could be used in the practice of the presentinvention. The present invention is in no way limited to the methods andmaterials described. In the event that one or more of the incorporatedliterature, patents, and similar materials differs from or contradictsthis application, including but not limited to defined terms, termusage, described techniques, or the like, this application controls.Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety.

The present disclosure relates to a compound4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamideof formula (I) or a pharmaceutically acceptable salt or hydrate thereof:

The compounds of the present disclosure have inhibitory activity againstprotein kinases. In one embodiment, the compounds are RAF inhibitorshaving inhibitory activity against at least one of the A-RAF, B-RAF andC-RAF isoforms. In one embodiment, the compounds are pan-RAF inhibitorshaving inhibitory activity against more than one of the A-RAF, B-RAF andC-RAF isoforms. In another embodiment, the inhibitory activity isagainst FMS. In another embodiment, the activity is against DDR1 andDDR2 kinases. In another embodiment, the activity is pan-RAF, is againstDDR1 and DDR2 kinases, and is against FMS.

In some embodiments, the compound of formula (I), and pharmaceuticallyacceptable salts and hydrates thereof of the present invention are insolid form.

In some embodiments, the compound of formula (I), and pharmaceuticallyacceptable salts and hydrates thereof of the present invention are insolution in a pharmaceutical carrier. In some such embodiments, theconcentration of formula (I) in solution is at least 0.01 mg/mL, atleast 0.05 mg/mL, at least 0.1 mg/mL, at least 0.5 mg/mL, at least 1mg/mL, at least 2 mg/mL, at least 3 mg/mL, at least 4 mg/mL or at least5 mg/mL.

In any of the various embodiments, the purity of the compound of formula(I), or a pharmaceutically acceptable salt or hydrate thereof, is atleast 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, atleast 99% or at least 99.5% as measured by HPLC. In any of the variousembodiments, the assay of the compound of formula (I), or apharmaceutically acceptable salt or hydrate thereof, is at least 95%, atleast 96%, at least 97%, at least 98%, at least 98.5%, at least 99% orat least 99.5% as measured by HPLC.

The compound of the present invention may also form a pharmaceuticallyacceptable salt. Such salt may be a pharmaceutically acceptable nontoxicacid addition salt containing anion, but not limited thereto. Forexample, the salt may include acid addition salts formed by inorganicacids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoricacid, hydrobromic acid, hydriodic acid, and others; organic carbonicacids such as tartaric acid, formic acid, citric acid, acetic acid,trichloroacetic acid, trifluoroacetic acid, gluconic acid, benzoic acid,lactic acid, fumaric acid, maleic acid, and others; and sulfonic acidssuch as methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonicacid, naphthalensulfonic acid, and others. In a specific embodiment, theacid addition salt is one formed by sulfuric acid, methanesulfonic acidor a hydrohalogenic acid (e.g., hydrochloric acid). In one embodiment,the formula (I) salt is the bis-hydrochloride salt.

In addition, hydrates of the compound of formula (I) are encompassedwithin the scope of the present invention.

The compound of the present invention may be obtained via General Scheme1 by using intermediates obtained in General Scheme 1 shown below, orstarting materials or intermediates which are commercially available,respectively. Further, mass analysis of the obtained4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidemay be performed by using MicroMass ZQ™ (Waters).

The pharmaceutical composition comprising, as an active ingredient,4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamideor salts or hydrates thereof may be used for prevention or treatment ofabnormal cell growth diseases caused by abnormal activation of one ormore protein kinases.

In addition, the present invention relates to a method for preventing ortreating a disease mediated by abnormal activation of one or moreprotein kinases, which method comprises administering the inventivecompound to a subject in need thereof. For example, the subject is amammal.

Examples of the protein kinases include ALK, AMPK, Aurora A, Aurora B,Aurora C, Axl, Blk, Bmx, BTK, CaMK, CDK2/cyclinE, CDK5/p25, CHK1, CK2,A-RAF, B-RAF, C-RAF, DDR1, DDR2, DMPK, EGFR1, Her2, Her4, EphA1, EphB1,FAK, FGFR2, FGFR3, FGFR4, Flt-1, Flt-3, Flt-4, FMS (CSF-1), Fyn, GSK3beta, HIPK1, IKK beta, IGFR-1R, IR, Itk, JAK2, JAK3, KDR, Kit, Lck, Lyn,MAPK1, MAPKAP-K2, MEK1, Met, MKK6, MLCK, NEK2, p70S6K, PAK2, PDGFRalpha, PDGFR beta, PDK1, Pim-1, PKA, PKB alpha, PKC alpha, Plk1, Ret,ROCK-I, Rsk1, SAPK 2a, SGK, Src, Syk, Tie-2, Tec, Trk or ZAP-70. In someembodiments, the one or more protein kinases include one or more ofA-RAF, B-RAF, C-RAF, DDR1, DDR2 and FMS. In some embodiments, the one ormore protein kinases include one or more of A-RAF, B-RAF and C-RAF. Insome embodiments, the one or more protein kinases include DDR1 and/orDDR2. In some embodiments, the protein kinase is FMS. The pharmaceuticalcomposition in accordance with the present invention has inhibitoryactivity against the above kinases.

Examples of the abnormal cell growth diseases caused by abnormalactivation of one or more protein kinases in which the inventivepharmaceutical composition is effective against include gastric cancer,lung cancer, liver cancer, colorectal cancer, small intestine cancer,pancreatic cancer, brain cancer, bone cancer, melanoma, breast cancer,sclerosing adenosis, uterine cancer, cervical cancer, head and neckcancer, esophagus cancer, thyroid cancer, parathyroid cancer, renalcancer, sarcoma, prostate cancer, urethral cancer, bladder cancer, bloodcancer, lymphoma, fibroadenoma, inflammation, diabetes, obesity,psoriasis, rheumatoid arthritis, hemangioma, acute and chronic kidneydisease, coronary restenosis, autoimmune diseases, asthma,neurodegenerative diseases, acute infection and ocular diseases causedby angiogenesis. In some embodiments, the cancer is melanoma or livercancer.

Pharmaceutical compositions of the present disclosure may comprise adrug selected from the group consisting of cell signal transductioninhibitors, mitosis inhibitors, alkylating agents, antimetabolites,antibiotics, growth factor inhibitors, cell cycle inhibitors,topoisomerase inhibitors, biological reaction modifiers, antihormonalagents, antiandrogens, cell differentiation/proliferation/survivalinhibitors, apoptosis inhibitors, inflammation inhibitors andP-glycoprotein inhibitors. In case where the inventive pharmaceuticalcomposition is developed into a formulation, it may be used incombination with said drug or developed into a combined formulation withsaid drug.

In some embodiments, the drug is a cell signal transduction inhibitor.In some such aspects the cell signal transduction inhibitor is a MEKinhibitor. Non-limiting examples of MEK inhibitor drugs include thefollowing. COTELLIC® (cobimetinib); (GDC-0973; Genentech);[3,4-difluoro-2-(2-fluoro-4-iodoanilino)phenyl]-[3-hydroxy-3-[(2S)-piperidin-2-yl]azetidin-1-yl]methanone;CAS #934660-93-2. GDC-0623 (Genentech);5-((2-fluoro-4-iodophenyl)amino)-N-(2-hydroxyethoxy)imidazo[1,5-A]pyridine-6-carboxamide;CAS #1168091-68-6. RO4987655 (Hoffman-La Roche); CH4987655;3,4-difluoro-2-(2-fluoro-4-iodoanilino)-N-(2-hydroxyethoxy)-5-[(3-oxooxazinan-2-yl)methyl]benzamide;CAS #874101-00-5. RO5126766 (Hoffman-La Roche);3-[[2-[(Methylaminosulfonyl)amino]-3-fluoropyridin-4-yl]methyl]-4-methyl-7-[(pyrimidin-2-yl)oxy]-2H-1-benzopyran-2-one;CAS #946128-88-7. Trametinib (GlaxoSmithKline); GSK1120212;N-[3-[3-cyclopropyl-5-[(2-fluoro-4-iodophenyl)amino]-3,4,6,7-tetrahydro-6,8-dimethyl-2,4,7-trioxopyrido[4,3-d]pyrimidin-1(2H)-yl]phenyl]-acetamide;CAS #871700-17-3. Pimasertib (Array BioPharma and AstraZeneca);AS-703026; Merck. Selumetinib; AZD6244;6-(4-bromo-2-chloroanilino)-7-fluoro-N-(2-hydroxyethoxy)-3-methylbenzimidazole-5-carboxamide;CAS #606143-52-6. Refametinib (Bayer); RDEA119; Bay 86-9766;N-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2S)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide;CAS #923032-37-5. PD184352 (Pfizer); CI-1040;(2-Chloro-4-iodophenylamino)-N-cyclopropylmethoxy-3,4-difluorobenzamide;CAS #212631-79-3. Binimetinib (Array BioPharma and Novartis); MEK162;5-((4-bromo-2-fluorophenyl)amino)-4-fluoro-N-(2-hydroxyethoxy)-1-methyl-1H-benzo[d]imidazole-6-carboxamide;CAS #606143-89-9. PD0325901 (Pfizer);N-[(2R)-2,3-Dihydroxypropoxy]-3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide;CAS #391210-10-9. AZD8330 (AstraZeneca);2-(2-fluoro-4-iodophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide;CAS #869357-68-6. TAK-733 (Millennium Pharmaceutical and TakedaPharmaceutical Company);(R)-3-(2,3-dihydroxypropyl)-6-fluoro-5-(2-fluoro-4-iodophenylamino)-8-methylpyrido[2,3-d]pyrimidine-4,7(3H,8H)-dione;CAS #1035555-63-5. WX-554 (Wilex, AG). HL-085 (Binjiang Pharma).Regorafenib (Bayer);4-(4-(3-(4-Chloro-3-(trifluoromethyl)phenyl)ureido)-3-fluorophenoxy)-N-methylpicolinamide;CAS #755037-03-7. RAF265 (Novartis); CHIR-265;1-methyl-5-[2-[5-(trifluoromethyl)-1H-imidazol-2-yl]pyridin-4-yl]oxy-N-[4-(trifluoromethyl)phenyl]benzimidazol-2-amine;CAS #927880-90-8. Lucitanib (Advan Chem); E3810;6-((7-((1-aminocyclopropyl)methoxy)-6-methoxyquinolin-4-yl)oxy)-N-methyl-1-naphthamide;CAS #1058137-23-7. Rebastanib (Deciphera Pharm); DCC2036;4-[4-[[[[3-(1,1-dimethylethyl)-1-(6-quinolinyl)-1H-pyrazol-5-yl]amino]carbonyl]amino]-3-fluorophenoxy]-N-methyl-2-pyridinecarboxamide;CAS #1020172-07-9. CK-127 (Chugai Pharma); RG7304;(E)-5-(2-benzylidene-1-methylhydrazinyl)pyridazin-3(2H)-one; CAS#213406-50-9. In some embodiments, the drug is cobimetinib.

The inventive pharmaceutical composition may comprise conventionalpharmaceutically acceptable excipients including carriers, diluents,adjuvants, additives and vehicles. The pharmaceutical composition may beformulated in accordance with conventional methods, and may be preparedin the form of oral formulations such as a tablet, pill, powder,capsule, syrup, an emulsion, a microemulsion and others or parenteralformulations such as intramuscular, intravenous or subcutaneousadministration.

When the pharmaceutical composition according to the present inventionis prepared as a formulation for oral administration, the carrier to beused may include, for instance and without limitation, cellulose,calcium silicate, corn starch, lactose, sucrose, dextrose, calciumphosphate, stearic acid, magnesium stearate, calcium stearate, gelatin,talc, surfactant, suspending agents, emulsifying agents, diluents, andcombinations thereof. Additionally, when the pharmaceutical compositionis prepared as a formulation for oral administration, the diluents to beused may include, for instance and without limitation, lactose,mannitol, saccharide, microcrystalline cellulose, cellulose derivative,corn starch, and combinations thereof. Formulations for oraladministration may also include, for instance and without limitation,polymers (for instance hydrophilic polymers such aspolyvinylpyrrolidone), antioxidants, preservatives, wetting agents,lubricating agents, glidants, processing aids, granulating agents,dispersing agents, colorants, and flavoring agents.

Compressed tablets can be prepared by compressing in a suitable machinethe active ingredient in a free-flowing form such as a powder orgranules, optionally mixed with a binder, lubricant, inert diluent,preservative, surface active or dispersing agent. Molded tablets can bemade by molding in a suitable machine a mixture of the powdered activeingredient moistened with an inert liquid diluent. The tablets canoptionally be coated or scored. Tablets can be uncoated or can be coatedby known techniques including microencapsulation to delay disintegrationand adsorption in the gastrointestinal tract and thereby provide asustained action over a longer period. For example, a time delaymaterial such as glyceryl monostearate or glyceryl distearate alone orwith a wax can be employed and optionally are formulated so as toprovide slow or controlled release of the active ingredient therefrom.

When the pharmaceutical composition according to the present inventionis prepared as a formulation for injections, the carrier to be used mayinclude, for instance and without limitation, water, saline, an aqueousglucose solution, an aqueous sugar-like solution, alcohols, glycols(e.g., polyethylene glycol 400), ethers, oils, fatty acids, fatty acidesters, glycerides, surfactants, suspending agents, emulsifying agents,and combinations thereof.

EXAMPLES Example 1: Preparation of Compound Formula (I)

Compound formula (I) may be prepared according to steps 1 to 4 asfollows.

The following abbreviations are used in Preparation Examples,Preparation Methods and Examples below.

Abbreviation Definition Fe Iron, powder CDMT4-Chloro-2,6-dimethoxy-1,3,5-triazine NMM 4-Methylmorpholine TEATriethylamine CHCl₃ Chloroform IPA Isopropyl alcohol EA Ethyl acetateHex n-Hexane EtOH Ethyl alcohol MeOH Methyl alcohol DMFN,N-dimethylformamide PW Purified water conc. HCl 35% Hydrochloric acidin water MgSO₄ Magnesium sulfate NaHCO₃ Sodium bicarbonate Brine Sodiumchloride in Water RBF Round-bottom flask NMR Nuclear Magnetic Resonance

The compound of formula (I) of the present invention may be prepared viaGeneral Scheme 1 by using intermediates obtained in General Scheme 1shown below, or starting materials or intermediates which arecommercially available, respectively.

The above reaction processes are exemplified in the following stepwisereactions.

Step 1: Preparation of 1-chloro-6-methylisoquinolin-5-amine

15.1 g of iron powder (0.27 mol, 3.0 eq.) and 0.3 mL concentrated HCl(0.005 mol, 0.05 eq.) were added to a 300 mL 1:1 mixed solution of ethylalcohol/purified water (15 v/w). The reaction mixture was heated up to90° C., followed by stirring for 1 hour. 20.0 g of1-chloro-6-methyl-5-nitroisoquinoline (0.09 mol, 1.0 eq.) was added tothe reaction mixture and stirred at reflux for 5 hours. The reactionmixture was filtered through a pad of Celite® (diatomaceous earth) underreduced pressure, and washed with a 100 mL 4:1 mixed solution ofchloroform/isopropyl alcohol (5 v/w). The filtrate was evaporated underreduced pressure, and the residue was dissolved in a 200 mL 4:1 mixedsolution of chloroform/isopropyl alcohol (10 v/w). The organic layer waswashed with 100 mL aqueous solution of sodium bicarbonate (5 v/w), and100 mL brine (5 v/w). The obtained organic layer was dried overanhydrous magnesium sulfate and concentrated under reduced pressure. Theconcentrated solid was dried under oven at 45° C. for 12 hours to obtainthe desired compound as a brown solid (14.3 g, 83%).

¹H-NMR spectrum (300 MHz, DMSO-d₄) δ 8.12 (m, 2H), 7.48 (q, 2H), 5.87(s, 2H), 2.28 (s, 3H).

Step 2: Preparation of4-amino-N-(1-chloro-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide

The 4-aminothieno[3,2-d]pyrimidine-7-carboxylic acid 28.5 g (0.15 mol,1.0 eq.) was dissolved in 300 mL N,N-Dimethylformamide (10 v/w), withsubsequent addition of 25.7 g of 4-chloro-2,6-dimethoxy-1,3,5-triazine(0.15 mol, 1.0 eq.) and 16.1 mL 4-methylmorpholine (0.15 mol, 1.0 eq.)at 20-30° C., followed by stirring for 20 minutes. 28.1 g of1-chloro-6-methylisoquinolin-5-amine (0.15 mol, 1.0 eq.) was then addedto the reaction mixture at 20-30° C., followed by stirring for 12 hours.To the reaction mixture was added 300 mL purified water (10 v/w) and thereaction mixture was stirred for 1 hour. The reaction mixture wasfiltered under reduced pressure, and washed with 90 mL purified water (3v/w). The obtained solid was dried under oven at 45° C. for 12 hours toobtain the desired compound as a brown solid (33.2 g, 61%).

¹H-NMR spectrum (300 MHz, DMSO-d₆) δ 11.69 (s, 1H), 8.95 (s, 1H), 8.65(s, 1H), 8.37 (m, 2H), 7.95 (s, 2H), 7.88 (m, 2H), 2.49 (s, 3H).

Step 3: Preparation of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride

20 g of4-amino-N-(1-chloro-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide(54.1 mmol, 1.0 eq.) and 17.4 g of 4-amino-2-chloro-3-fluorophenol(108.2 mmol, 2.0 eq.) were added to a sealed tube and 200 mL isopropylalcohol (10 v/w) and 13.5 mL concentrated HCl (162.2 mmol, 3.0 eq.) wereadded to the reaction mixture at 20-25° C. The reaction mixture washeated up to 130° C., followed by stirring for 12 hours. The reactionsolution was cooled to 20-30° C. The solid was filtered under reducedpressure, followed by washing with 50 mL isopropyl alcohol 100 mL (5v/w) to obtain the desired compound as a dark brown solid (23.5 g, 77%).

Step 4: Preparation of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide

23.5 g of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride (41.4 mmol, 1.0 eq.) was added to a 1 L round-bottomflask and 470 mL methyl alcohol (20 v/w) was added. 12.7 mLtriethylamine (91.0 mmol, 2.2 eq.) was added to the reaction mixture at20-25° C., followed by stirring for 2 hours. The solid was filteredunder reduced pressure, followed by washing with 100 mL methyl alcohol(5 v/w). The filtered solid was dried under oven at 45° C. for 12 hoursto obtain the desired compound as brown solid (11.7 g, 57%).

¹H-NMR spectrum (300 MHz, DMSO-d₆) δ 11.53 (s, 1H), 10.48 (br, 1H), 8.97(s, 1H), 8.92 (d, 1H), 8.50 (s, 1H), 8.32 (d, 1H), 7.94 (s, 2H), 7.79(d, 1H), 7.55 (d, 1H), 7.20 (t, 1H), 7.04 (d, 1H), 6.82 (d, 1H), 2.41(s, 3H).

Example 2: Preparation of Compound Formula (I) Bis-Hydrochloride SaltStep 1: Preparation of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride

11.7 g of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamide(23.6 mmol, 1.0 eq.) was added to a 500 mL round-bottom flask and 240 mLof 80% ethyl alcohol (20 v/w) was added, followed by the addition of 5.9mL of concentrated HCl (70.9 mmol, 3.0 eq.) to the reaction mixture at20-25° C. The reaction mixture was heated up to 90° C., followed bystirring for 2 hours. The reaction solution was cooled to 20-30° C. Thesolid was filtered under reduced pressure, followed by washing with 58.5mL ethyl alcohol (5 v/w). The filtered solid was dried in an oven at 45°C. for 12 hours to obtain the desired compound as brown solid (10.9 g,81%).

Step 2: Refinement of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride

10.9 g of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride was added to a 3 L round-bottom flask and 2 L of methylalcohol (200 v/w) was added at 20-25° C. The reaction mixture was heatedup to 50° C., followed by stirring for 30 min. The reaction mixture wascooled to 20-30° C. 1.1 g of activated carbon was added, followed bystirring for 3 hours. The reaction mixture was filtered through a pad ofCelite under reduced pressure, followed by washing with 100 mL of methylalcohol (10 v/w). The filtrate was filtered (using membrane filterpaper) under reduced pressure, followed by washing with 100 mL of methylalcohol (10 v/w). The filtrate was evaporated under reduced pressure. Tothe residue was added methyl alcohol (200 mL), followed by stirring for2 hours. The solid was filtered under reduced pressure, followed bywashing with 50 mL of methyl alcohol (5 v/w). The filtered solid wasdried in an oven at 45° C. for 12 hours to obtain the desired compoundas a pale brown solid (7.7 g, 71%).

Step 3: Refinement of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride

7.7 g of4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidedihydrochloride (13.6 mmol, 1.0 eq.) was added to a 250 mL round-bottomflask and 150 mL of 80% ethyl alcohol (20 v/w) and 2.3 mL ofconcentrated HCl (27.1 mmol, 2.0 eq.) were added to the reaction mixtureat 20-25° C. The reaction mixture was heated up to 90° C., followed bystirring for 1 hour. The reaction solution was cooled to 20-30° C. Thesolid was filtered under reduced pressure, followed by washing with 38.5mL of ethyl alcohol (5 v/w). To the resulting solid was added 385 mL ofmethyl alcohol (50 v/w). The reaction mixture was heated up to 40° C.,followed by stirring for 3 hours. The solid was filtered under reducedpressure, followed by washing with 38.5 mL of methyl alcohol (5 v/w).The filtered solid was dried at 45° C. for 12 hours. The solid compoundwas then dried at 25° C., 90% RH for 12 hours. The hygroscopic solid wasthen dried at room temperature for 6 hours to obtain the desiredcompound as an off-white powder (4.8 g, 62%).

Purity: 97.0% by HPLC

Assay: 96.8% by HPLC

Moisture: 4.7%

1H-NMR spectrum (300 MHz, DMSO-d6) δ 11.60 (s, 1H), 11.42 (s, 1H), 9.47(s, 1H), 8.88 (d, 1H), 8.62 (s, 1H), 7.88 (d, 1H), 7.56 (d, 1H), 7.42(t, 1H), 7.32 (d, 1H), 7.10 (d, 1H), 2.50 (s, 3H).

Experimental Example 3: Evaluation of RAF Kinase Activity

Formula (I) bis-hydrochloride salt prepared in Example 2 (hereinafterreferred to as “Compound 2.HCl”) was tested for inhibitory activityagainst three subtypes of RAF, i.e., RAF1 (C-RAF)^(Y34D/N341D), B-RAFwild type and B-RAF^(V600E) using Kinase Profiling Service (ThermoFisher Scientific, previously Invitrogen, U.S.A) according to themanufacturer's instructions. The levels of enzymatic inhibition of thecompound were calculated as percent inhibition at variousconcentrations. Based on percent inhibition, dose-response curves wereplotted using GraphPad Prism software. The IC₅₀ values of Compound.2HClagainst C-RAFY^(Y340D/Y341D), B-RAF^(WT) and B-RAF^(V600E) are listed inTable 1 where ZELBORAF® (vemurafenib, PLX-4032, Roche) was used as acontrol.

TABLE 1 B-RAF^(WT) B-RAF^(V600E) C-RAF^(Y340D/Y341D) Example (IC₅₀, nM)(IC₅₀, nM) (IC₅₀, nM) Control 111 107 138 Compound•2HCl 392 30 21

Experimental Example 4: Evaluation of Inhibition on Cell Growth ofN-RAS^(Q61K) Mutant Cell Line SK-MEL-30 (Human Melanoma Cells)

The inventive compound was tested for inhibitory activities onproliferation aberrant cells as follows.

N-RAS^(Q61K) mutant SK-MEL-30 melanoma cell line (ACC-151) was obtainedfrom DSMZ (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH,Germany). SK-MEL30 cells express wildtype B-RAF and mutant N-RAS (Q61K).SK-MEL-30 cells were incubated in a RPMI medium supplemented with 10%FBS and 1% penicillin/streptomycin (Gibco BRL) under 37° C., 5% CO2 and95% air. Cells were seeded in 96-well plates at a density of 5,000cells/well, and cultured for 18 hours or more. Then cells were treatedwith 0.1-10 μM (1/10 serial dilution) of test compounds, and incubatedfor 72 hours.

To evaluate cell viabilities, SK-MEL-30 cell lines were fixed with 10%TCA (trichloroacetic acid), stained with SRB (sulforhodamine B), and theabsorbance was measured at 540 nm. Then, GI₅₀, i.e., the concentrationof drug to cause 50% reduction in proliferation of cancer cells, wascalculated therefrom. The growth rates of cancer cells were calculatedby Equation 1 or 2.

[(Ti−Tz)/(C−Tz)]×100 (for Ti≥Tz)  Equation 1

[(Ti−Tz)/Tz]×100 (for Ti<Tz)  Equation 2

In Equation 1 and 2, ‘Tz’ refers to the density of untreated cells,which is the absorbance in 0% cell growth groups. ‘C’ refers to thedensity of cells cultured by adding only medium, and ‘Ti’ refers to thedensity of cells treated with test compounds.

GI₅₀ value is the concentration of a test compound when the value ofEquation 1 is 50, which indicates the concentration of the test compoundneeded to reduce the growth of cancer cells to 50%. On each measurement,test compounds were compared with a vemurafenib (PLX-4032) control. TheGI₅₀ values of each compound were measured and are shown in Table 2.

TABLE 2 SK-MEL-30 Example (GI₅₀, nM) Control >10,000 Compound•2HCl 231

As evidenced above, the inventive compound,4-amino-N-(1-((3-chloro-2-fluoro-4-hydroxyphenyl)amino)-6-methylisoquinolin-5-yl)thieno[3,2-d]pyrimidine-7-carboxamidehaving inhibitory activity for protein kinases, can effectively inhibitvarious protein kinases including RAF, and thus can be used, singly orin combination, for prevention and treatment of diseases associated withaberrant cell growth which are caused by mutation or overexpression ofRAS protein or overactivation of its associated protein kinases.

What is claimed is:
 1. A method for preventing or treating a diseasemediated by abnormal activation of one or more protein kinases, themethod comprising administering an effective amount of the compound offormula (I), or a pharmaceutically acceptable salt or hydrate thereof toa mammal in need thereof.
 2. The method of claim 1, wherein at least oneof said one or more protein kinases is selected from: ALK, AMPK, AuroraA, Aurora B, Aurora C, Axl, Blk, Bmx, BTK, CaMK, CDK2/cyclinE, CDK5/p25,CHK1, CK2, A-RAF, B-RAF, C-RAF, DDR1, DDR2, DMPK, EGFR1, Her2, Her4,EphA1, EphB1, FAK, FGFR2, FGFR3, FGFR4, Flt-1, Flt-3, Flt-4, FMS(CSF-1), Fyn, GSK3 beta, HIPK1, IKK beta, IGFR-1R, IR, Itk, JAK2, JAK3,KDR, Kit, Lck, Lyn, MAPK1, MAPKAP-K2, MEK1, Met, MKK6, MLCK, NEK2,p70S6K, PAK2, PDGFR alpha, PDGFR beta, PDK1, Pim-1, PKA, PKB alpha, PKCalpha, Plk1, Ret, ROCK-I, Rsk1, SAPK 2a, SGK, Src, Syk, Tie-2, Tec, Trkand ZAP-70.
 3. The method of claim 2 wherein at least one of said one ormore protein kinases is selected from: A-RAF, B-RAF, C-RAF, DDR1, DDR2and FMS.
 4. The method of claim 1, wherein said disease is selected fromthe group consisting of: gastric cancer, lung cancer, liver cancer,colorectal cancer, small intestine cancer, pancreatic cancer, braincancer, bone cancer, melanoma, breast cancer, sclerosing adenosis,uterine cancer, cervical cancer, head and neck cancer, esophagus cancer,thyroid cancer, parathyroid cancer, renal cancer, sarcoma, prostatecancer, urethral cancer, bladder cancer, blood cancer, lymphoma,fibroadenoma, inflammation, diabetes, obesity, psoriasis, rheumatoidarthritis, hemangioma, acute and chronic kidney disease, coronaryrestenosis, autoimmune diseases, asthma, neurodegenerative diseases,acute infection and ocular diseases caused by angiogenesis.
 5. Themethod of claim 1, which is used in combination or developed into acombined formulation with a co-drug selected from the group consistingof cell signal transduction inhibitors, mitosis inhibitors, alkylatingagents, antimetabolites, antibiotics, growth factor inhibitors, cellcycle inhibitors, topoisomerase inhibitors, biological reactionmodifiers, antihormonal agents, antiandrogens, celldifferentiation/proliferation/survival inhibitors, apoptosis inhibitors,inflammation inhibitors and P-glycoprotein inhibitors.
 6. The method ofclaim 5, wherein the drug is a signal transduction inhibitor that is aMEK inhibitor.